Primer sequences used for qRT-PCR analysis.

The rise of multidrug-resistant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa has driven the search for novel antimicrobial agents with enhanced efficacy and reduced toxicity. Tachyplesin I (TP-I), a β-sheet antimicrobial peptide isolated from horseshoe crab hemocytes, is known for its broad-spectrum activity but is limited by the presence of cysteine-rich disulfide bonds. In this study, we evaluated two synthetic analogs: CDT (Cysteine-Deleted Tachyplesin I) and CRDT (Cysteine- and Arginine-Deleted Tachyplesin Analog), designed to simplify the structure and reduce production cost while maintaining or enhancing bioactivity. The antimicrobial efficacy of CDT and CRDT was assessed against S. aureus and P. aeruginosa through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. CRDT demonstrated potent antimicrobial activity, with enhanced membrane-disruptive effects visualized via scanning electron microscopy (SEM), especially in P. aeruginosa. Molecular docking revealed strong binding affinities between CRDT and key QS regulators—SarA in S. aureus, and LasR in P. aeruginosa—supporting its ability to interfere with bacterial communication systems, while qRT-PCR analysis showed significant downregulation of QS-related genes (agrA, sarA, hla, algD and pelA). These findings suggest that CRDT not only exhibits direct bactericidal activity but also interferes with QS-mediated communication, making it a promising candidate for the development of dual-action antimicrobial therapeutics targeting both bacterial viability and virulence.以金黄色葡萄球菌（Staphylococcus aureus）、铜绿假单胞菌（Pseudomonas aeruginosa）为代表的多重耐药病原体的出现，促使科研人员寻求兼具增效与减毒特性的新型抗菌剂。鲎素I（Tachyplesin I，TP-I）是一种从鲎血细胞中分离得到的β折叠型抗菌肽，具备广谱抗菌活性，但因富含半胱氨酸的二硫键结构存在应用局限。本研究评估了两种合成类似物：半胱氨酸缺失型鲎素I（Cysteine-Deleted Tachyplesin I，CDT）与半胱氨酸-精氨酸双缺失型鲎素类似物（Cysteine- and Arginine-Deleted Tachyplesin Analog，CRDT），二者旨在简化结构、降低生产成本的同时保留或增强生物活性。通过最低抑菌浓度（minimum inhibitory concentration，MIC）与最低杀菌浓度（minimum bactericidal concentration，MBC）实验，评估了CDT与CRDT对金黄色葡萄球菌和铜绿假单胞菌的抗菌效果。结果显示，CRDT展现出强效抗菌活性，扫描电子显微镜（scanning electron microscopy，SEM）可视化观察表明其具备更强的膜破坏作用，尤其针对铜绿假单胞菌。分子对接实验证实，CRDT与关键群体感应（Quorum Sensing，QS）调控因子——金黄色葡萄球菌中的SarA蛋白以及铜绿假单胞菌中的LasR蛋白——具有较强结合亲和力，提示其可干扰细菌群体感应通信系统；实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction，qRT-PCR）分析显示，CRDT可显著下调QS相关基因（agrA、sarA、hla、algD及pelA）的表达水平。上述研究结果表明，CRDT不仅可直接发挥杀菌活性，还能干扰QS介导的细菌通信通路，因此是一种兼具靶向细菌存活与毒力双重作用的新型抗菌治疗候选药物。